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[Preparation of rabbit anti-STAT5 antibody and its application in breast cancer diagnosis]
Jing Chen1, Bao-en Shan, Xing Chen
1Clinical Laboratory, Bethune International Peace Hospital, Shijiazhuang 050082, China.
Aim:
To prepare and purify rabbit antibody against STAT5 and evaluate its application in breast cancer diagnosis.
Methods:
The rabbits were immunized with recombinant mouse STAT5 protein for preparing antibody against STAT5. The purity and specificity of the antibody were analyzed by Western blot. The proteins expressed in human breast cancer cell lines and normal breast tissue and breast cancer tissues were analyzed by Western blot and immunohistochemical staining with the anti-STAT5 antibody.
Results:
The titer of anti-STAT5 antibody in the serum was 1:204 800 (A(450)=1.07) at 6 weeks after being immunized with STAT5. The SDS-PAGE and Western blot analysis showed that there was a obvious protein band with M(r) 90 000-95 000, confirming the specificity of obtained anti-STAT5 antibody. The expression of STAT5 could be detected with Western blot in both human breast cancer cell lines MCF-7 and MDA-MB-231. Immunohistochemical staining indicated that STAT5 was expressed in cytoplasm of normal breast tissues while in cytoplasm and nucleolus of breast cancer tissues.
Conclusion:
The rabbit anti-STAT5 antibody with high titer and specificity has been prepared. The STAT5 is expressed in cytoplasm of normal breast tissue, and in nucleolus of breast cancer tissue. The preparation of anti-STAT5 antibody provides an important reagent for clinical diagnosis of breast cancer.
